Synthesis and evaluation of mycophenolic acid derivatives as potential anti-Toxoplasma gondii agents

Nineteen mycophenolic acid (MPA) derivatives were designed and synthesized, and their anti-Toxoplasma activity evaluated for the first time. Among them, N-propylimidazole-modified compound E5 demonstrated the strongest activity, and the IC50 against HFF-1 (Human Foreskin Fibroblasts-1) cells following infection with T. gondii is 80.9 μM (MPA-211.5 μM) and its selectivity index is 2.2 (MPA-1.2). In vivo experiments, E5 can significantly inhibit the proliferation of tachyzoites in the abdominal cavity of mice acutely infected with T. gondii (inhibition rates 46.7%), and this inhibitory effect was greater than that of spiramycin (inhibition rates 31.3%) and MPA (inhibition rates 15.9%), this indicated that E5 had significant protective effects on the host during acute Toxoplasma infection. In addition, E5 can reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of infected mice, significantly increase the level of glutathione (GSH) in the liver, and significantly reduce the level of malondialdehyde (MDA), indicating that it has a hepatoprotective effect against T. gondii infection. Similarly, E5 can relieve hepatomegaly and splenomegaly induced by acute Toxoplasma infection. Spiramycin aggravated appetite loss in infected mice, while E5 did not. In summary, the results indicated that E5 has potential as a candidate anti-T. gondii drug.


Introduction
Toxoplasma gondii is an obligate intracellular parasite with a wide variety of hosts, including humans, livestock and marine mammals, and can cause zoonotic toxoplasmosis [1]. Following infection, individuals with normal immune function show no obvious symptoms, while those with insufficient or low immune function may display extensive pathological damage or even death. In pregnant females, infection can lead to miscarriage, premature delivery, stillbirth and teratogenesis [2,3]. Toxoplasma gondii can also infect domestic animals, such as pigs, cattle and sheep, and often infects companion animals, such as dogs, cats and rabbits. The pathogen also causes huge economic losses to the livestock industry, and T. gondii is detrimental to food safety and human health [4,5]. At present, the main treatment for toxoplasmosis is still drug therapy, mainly including pyrimethamine, sulfadiazine, spiramycin and the combined use of sulfadiazine and pyrimethamine. Although there has been some success with anti-Toxoplasma drugs, many problems remain. For example, the use of anti-Toxoplasma drugs can lead to adverse reactions, such as allergies, bone marrow suppression and increased risk of liver and kidney complications. In addition, these drugs are not suitable for use during pregnancy [6][7][8]. Therefore, there is an urgent need for the development of anti-Toxoplasma drugs with high efficiency and low toxicity.
The immunosuppressant mycophenolate mofetil has been used widely to prevent and treat the acute rejection of transplanted organs. Mycophenolate mofetil exerts immunosuppressive activity via conversion into mycophenolic acid (MPA, Fig. 1) in the body [9][10][11]. MPA is a meroterpenoid consisting of an acetate-derived phthalide nucleus and a terpene-derived side chain, which possesses a range of biological properties, including antifungal [12], antiviral [13], anti-inflammatory [14] and antitumor [15] properties. In addition, MPA is a potent non-competitive inhibitor of inosine nucleotide dehydrogenase inosine monophosphate dehydrogenase (IMPDH). However, IMPDH is essential for the purine metabolism of Toxoplasma gondii. More importantly, MPA has been used extensively in strategies for the generation of genetically modified strains of Toxoplasma gondii [16]. To our knowledge, there have been no reports on the activity of MPA derivatives against T. gondii. Therefore, in this study, we modified the structure of MPA to obtain high-efficiency and low-toxicity anti-Toxoplasma drugs.
Studies have reported that the lactone ring and aromatic methyl group in the structure of MPA are essential to its activity [17]. Therefore, in this study, these two groups were retained, and the carboxyl position of the structure was modified to introduce nitrogen-containing pharmacophores and convert it into an amide. The amide group can be used as a hydrogen donor, reducing the sensitivity of enzymatic hydrolysis and improving the stability of the compound in vivo and in vitro [18]. Amino acids are the basic biological and functional units of proteins; and they play a vital role in human metabolism and exert a wide range of biological activities, including antibacterial [19,20], anticancer [21], antituberculosis [22], anti-inflammatory [23,24] and anti-parasitic [25,26] activities. In addition, they improve both the solubility and bioavailability of the drugs [27][28][29]. Studies have reported that nitrogen-containing heterocycles, such as benzylamine, pyrrolidine, piperazine, morpholine and imidazole, are an important class of active fragments against T. gondii [30][31][32]. In addition, active fragments with these functions also include benzylamine and fatty amines [29]. Therefore, we designed and synthesized five series of MPA derivatives and evaluated their anti-Toxoplasma activity. Subsequently, we selected the most active derivative E5, at the cellular level for subsequent in vivo activity studies to find highly efficient and low-toxicity MPA derivatives with anti-Toxoplasma activity.

Chemistry
The synthetic route for targets is shown in Scheme 1. MPA as a starting material and all target compounds were obtained by an amide condensation reaction with different amino compounds catalysed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and Biphenyl-4-amidoxime (HOBt) in anhydrous CH 2 Cl 2 at 0°C [33,34]. TLC was used to monitor the reactions, and the final products were purified using silica gel chromatography to obtain the target compounds. Among them, compounds A1, A3, A4, C1, C2, E3 have been reported [35,36]. All compounds were confirmed by 1 H NMR, 13 C NMR and high-resolution mass spectrometry.

Biological evaluation
In vitro anti-T. gondii activity and structure-activity relationship (SAR) study The thiazolyl blue (MTT) colorimetric method was used to determine the cytotoxicity and in vitro anti-tachyzoites activity (T. gondii, RH strain) of all compounds at different concentrations (10-1000 μM) in an activity test in host cells (Human Foreskin Fibroblasts-1 cells) [37].
The in vitro anti-Toxoplasma activity is shown in Table 1. IC 50 a is the half-inhibitory concentration of the compound against HFF-1 cells, which reflects its cytotoxicity. IC 50 b is the half-inhibitory concentration of the compound against HFF-1 cells following infection with T. gondii; this value reflects the anti-T. gondii activity of the compound. The ) provides a screening reference for subsequent in vivo experiments [18]. In this experiment, MPA was used as the lead compound, different types of pharmacophores were introduced, and five series of compounds were designed and synthesised with spiramycin being used as the positive control drug. The anti-Toxoplasma activity of MPA in vitro was greater than that of spiramycin, which has strong anti-Toxoplasma potential. In the compound A series, different substituted aromatic hydrocarbons were introduced into the MPA. The in vitro anti-Toxoplasma activity of all compounds was lower than that of lead, however, it is worth noting that most of the compounds showed very low cytotoxicity, except for A1, the selectivity of all compounds has been improved, and their SI value was higher than that of MPA and the positive control drugs. In this series, the preliminary structure-activity relationship demonstrated that the introduction of electron-withdrawing groups or electron-donating groups on the benzene ring, or appropriately extended the length of the aliphatic chain, increased the in vitro anti-Toxoplasma activity. In the compound B series, different alkanes were introduced into the MPA. Compared with that of the lead, all compounds demonstrated reduced in vitro anti-Toxoplasma activity and a lower SI value. This suggests that the introduction of alkanes on MPA decreased the effectiveness against T. gondii. The compound C series was endowed with different amino acid methyl ester fragments into the lead. In this group, compound C1 and lead exhibited similar in vitro anti-Toxoplasma activity. The SI value of compound C2 was 1.6, which was better than that of the lead and the positive control drugs. In the compound D series, four different amine compounds were connected to MPA. Unfortunately, the in vitro anti-Toxoplasma activity and SI values of all compounds were not ideal. The compound E series are connected to different heterocyclic compounds, and the in vitro anti-Toxoplasma activity of compounds E3, E4 and E5 were improved. Compound E5 obtained by the introduction of an N-propylimidazole fragment to the lead, and this compound presented the strongest in vitro anti-Toxoplasma activity among all series, and the IC 50 against HFF-1 cells following infection with T. gondii is 80.9 μM. In addition, it has the highest selectivity in this article (SI: 2.2), its IC 50 and SI values were significantly better than those of the positive control drug (IC 50 : 233.4 μM; SI:1.2) and mycophenolic acid (IC 50 : 211.5 μM; SI:1.2), which indicates that it has the highest selectivity index while maintaining in vitro anti-Toxoplasma activity and has some research value. Therefore, in this experiment, compound E5 was selected for the subsequent evaluation of anti-T. gondii activity in vivo.

Effect of compounds E5 on the inhibition of tachyzoites in vivo
The number of T. gondii tachyzoites in the abdominal cavity of mice reflects the therapeutic effects of a compound [38]. As shown in Table 2 and Fig. 2, compared with that of the model group, after treatment with 100 mg/kg of different compounds, the inhibition of intraperitoneal tachyzoites in mice in the MPA treatment group was only 15.9%. Interestingly, intraperitoneal tachyzoite inhibition in the E5 treatment groups was 46.7%, respectively, and there were significant differences compared with the model group (p < 0.001). Therefore, compound E5 significantly reduced the number of T. gondii tachyzoites in the abdominal cavity of Kunming mice and their activity exceeded that of the positive control spiramycin, indicating that E5 is effective against acute T. gondii infection. The protective effect on the host during infection was significant.

Liver and spleen indices
A large number of studies have shown that the liver is the main diseased organ in mice, which infected with acute infection models by intraperitoneal administration of toxoplasma gondii virulent strains. Liver is an important site for T. gondii replication, which leads to hepatic pathological Each value is expressed as the mean ± standard deviation (SD) (n = 3) IC 50 a is the half-inhibitory concentration of the compound against host cells, HFF-1 IC 50 b is the half-inhibitory concentration of the compound against HFF-1 following infection with T. gondii c SI: Selectivity index, a measure of efficacy, calculated by IC 50 in HFF-1 cells/IC 50 in T. gondii  changes such as hepatomegaly, hepatitis, granuloma, necrosis, biliary jaundice and cirrhosis in infected hosts [39]. Hepatic function and metabolism reduce when hepatocytes are damaged. Therefore incomplete metabolized substances are produced in liver, resulting in an increase on the liver index [40]. The level of the liver index reflects the degree of liver damage [39]. Moreover, the spleen is an important immune organ, which plays a significant role in the immune response, including the response to antigenic substances in the blood. Both humoral and cellular immunity can induce changes in the organizational structure of the spleen. To a certain extent, an increase in the spleen index reflects changes in spleen structure and function [41,42]. Therefore, the liver and spleen indices can be used to evaluate the protective effects of drugs on organs. As shown in Fig. 3, compared with those in the normal group, the liver and spleen indices of the toxo group increased, indicating that T. gondii induces liver and spleen damage in mice. Compound E5 was able to alleviate hepatomegaly and splenomegaly caused by acute Toxoplasma infection.

ALT and AST in serum
The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum are used to evaluate liver function. In this experiment, the liver toxicity of each compound was evaluated by determining the levels of ALT and AST in the serum of mice [43]. As shown in Fig. 4, compared with those in the normal group, the ALT and AST levels in the toxo group were significantly increased, and this was alleviated in the treatment group. Notably, compound E5 was able to reduce the levels of ALT and AST in the serum of infected mice. These results show that compound E5 is effective in treating T. gondii infection and preventing hepatotoxicity caused by T. gondii infection.

GSH and MDA
Glutathione (GSH) is a small-molecule peptide composed of three amino acids that is involved in detoxification, its main functions are to scavenge free radicals, resist oxidation and resist aging [44]. According to the literature, Toxoplasma gondii infection can cause oxidative in stress and immune suppression in blood donors [45]. During the infection of Toxoplasma gondii, the production of reactive oxygen species (ROS) increases, and the excessive production of ROS can cause cell and tissue damage [46,47]. GSH can protect cells from oxidative damage by eliminating superoxide anion free radicals. And its levels in mice exposed to Toxoplasma gondii are significantly reduced  [48,49]. As shown in Fig. 5A, compared with the normal group, the GSH content of the toxo group was significantly reduced, indicating that T. gondii infection can decrease the GSH content in mice. Interestingly, compared with that of the model group, GSH content was increased in mice in the treatment group, and the activity of compound E5 was greater than that of the positive control spiramycin and the lead compound MPA. This indicates that compound E5 is effective at inhibiting the growth of T. gondii in mice and has a strong antioxidant effect. Malondialdehyde (MDA), one major product of lipid peroxidation, significantly increased in mice exposed to T. gondi [50]. As shown in Fig. 5B, compared with that of the normal group, the MDA content in the toxo group significantly increased as we predicted, while spiramycin, MPA and E5 significantly reduced MDA content. Among them, compound E5 had the same curative effect as spiramycin and was better than lead MPA.

Effects of compounds on the weight of mice
To further evaluate the effects of the test compounds on mice, the weight change of animals during treatment was recorded. As shown in Fig. 6, compared with the normal group, mice in the toxo group lost their appetite due to T. gondii infection, resulting in a sharp decline in body weight. However, this was exacerbated by spiramycin and MPA. Unexpectedly, compound E5 did not result in weight loss, moreover alleviated the loss of appetite in mice.

Conclusions
In this study, 19 MPA derivatives in five series were designed and synthesised, and their anti-Toxoplasma activity was reported for the first time. The in vitro results demonstrated that E5 was the greatest potential and was superior to MPA and spiramycin. The results of further in vivo experiments showed that E5 exerted an inhibitory effect on T. gondii, a protective effect on the liver, and could alleviate splenomegaly in infected mice with limited side effects. These findings suggest that E5 is a potential candidate anti-T. gondii drug.

Experimental section
Mycophenolic acid is a natural compound, purchased from Chengdu Refines Biotechnology Co., Ltd., with a purity of 98%. The organic reagents required for the experiment are all analytically pure, and other chemicals are purchased from Aladdin reagent. The obtained products were monitored by thin-layer chromatography to check the progress of the reaction, and separation was carried out using a chromatography column. The melting point of the target product was measured in an open capillary (the temperature is not corrected); 1 H-NMR and 13 C-NMR used the chemical shift of TMS as the zero point, measured by AV-300 nuclear magnetic resonance instrument; the highresolution mass spectrum was measured by ESI mass spectrometer. Values are expressed as mean ± S.D. (n = 6). *p < 0.05, **p < 0.01 and ***p < 0.001 compared with normal; # p < 0.05, ## p < 0.01 and ### p < 0.001 compared with Toxo Fig. 6 Effect of the test compounds on the body weights of Toxoplasma gondii-infected KM mice. Values are expressed as mean ± S. D. (n = 6). *p < 0.05 and **p < 0.01 compared with normal General procedure for the reaction of mycophenolic acid with different intermediates containing-amino Mycophenolic acid (64 mg, 0.2 mmol), different amine compounds (0.24 mmol), carboxylic mixture 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (114.7 mg, 0.6 mmol) and catalyst Biphenyl-4-amidoxime (HOBt) (84.8 mg, 0.4 mmol) were taken in a 25 ml round bottom flask, and 5 ml dichloromethane as solvent, stirred at 0°C for 4-6 h, progress of reaction was confirmed by TLC, the final products were purified using silica gel chromatography, and all the target compounds were obtained, which are white powder in appearance, and the yield is 48-79%.

The liver and spleen index assay
After the mice of each group were sacrificed by neck dissection, they were dissected, the liver and kidney of the mice were separated, and the weight of the liver and spleen of each mouse was weighed and recorded, and then the liver and spleen index was obtained. The dissected liver and spleen were stored at low temperature, and the unused internal organs were temporarily stored in a refrigerator at −80°C.

Determination of ALT and AST in serum
The determination of AST and ALT in serum is based on Lai's method. An appropriate amount of serum was mixed with five times the amount of ALT or AST matrix buffer, then it was placed at 37°C for 30 min and 1 mmol/L 2,4dinitrophenylhydrazine solution equal in volume was added to the matrix buffer and mixed well, after 20 min, 0.4 mol/L sodium hydroxide solution was added and placed at room temperature for 5 min. Adjusting the zero with distilled water at a wavelength of 505 nm, the absorbance of each sample was read and calculated, and obtained the concentration of ALT and AST in the serum (U/L) through the standard curve [51]. ALT matrix buffer: 1.79 g of DL-alanine and 29.2 mg of α-ketoglutarate were accurately weighed, and dissolved using an appropriate amount of 0.1 mol/L phosphate buffer. After that, 1 mol/L of sodium hydroxide solution was added to adjust the pH to 7.4. Finally, additional phosphate buffer was added to adjust the volume to 100 ml to obtain the ALT matrix buffer (stored at 4°C).
AST matrix buffer: 24.2 mg α-ketoglutarate and 2.66 g DL-aspartic acid were accurately weighed, and dissolved using appropriate amount of 0.1 mo/L phosphate buffer. After that, 1 mol/L of sodium hydroxide solution was added to adjust the pH to 7.4. Finally, Finally, additional phosphate buffer was added to adjust the volume to 100 ml to obtain the AST matrix buffer (stored at 4°C).

Determination of GSH and MDA in liver homogenate
Preparation of liver homogenate: A certain amount of liver tissue was washed using pre-cooled physiological saline, and then an electric homogeniser was used to prepare a 20% tissue homogenate in 0.85% normal saline after drying. Followed by centrifugation (4000 rpm, 10 min), the supernatant was taken as a liquid reserve. The whole process should be carried out at low temperature as much as possible.
Protein content determination: The protein content of each group of mouse liver homogenate was tested according to the BCA protein concentration determination kit (Shanghai Biyuntian Biotechnology Co., Ltd.).
GSH determination: An appropriate amount of liver homogenate supernatant was mixed according to the supernatant: 20% trichloroacetic acid = 2:1, followed by centrifugation (4000 rpm, 10 min). Subsequently, the prepared 0.3 mol/L disodium hydrogen phosphate buffer and 0.04% DTNB reagent were added in sequence and mixed thoroughly, and then the absorbance value was measured at 412 nm. In the experiment, the GSH standard curve was drawn with glutathione standard substance and the regression equation was obtained. Substituting the measured absorbance value of the experimental sample into the regression equation, the concentration of GSH in each sample solution can be calculated. Finally, the GSH content in the liver homogenate (mg/gprot) = the measured GSH concentration in the sample solution (mg/ml)/the liver homogenate protein concentration (gprot/ml) [52].
MDA determination: MDA is determined by the thiobarbituric acid method. A certain amount of liver homogenate supernatant was mixed with 0.5% thiobarbituric acid, and then it was boiled in a water bath for 1 h, centrifuged after cooling (6000 rpm, 10 min). Moreover, the upper pink clear liquid was measured absorbance value at 532 nm. In addition, 1,1,2,2-tetraethoxypropane was used as a standard sample to draw a standard curve, and the absorbance value of the experimental sample was substituted into the standard curve to obtain the MDA concentration of each sample solution. Finally, the MDA content in the liver homogenate (μmol/gprot) = the measured MDA concentration in the sample solution (μmol/ml)/ the liver homogenate protein concentration (gprot/ml) [53].